title: Rapid prenatal diagnosis of common beta-thalassemia mutations in Southeast Asia using pyrosequencing.
abstract: OBJECTIVE
  Current methods of prenatal diagnosis to detect beta-thalassemia are Sanger sequencing and reverse dot blot. These methods are time-consuming and can prolong assay turnaround time. We aim to develop a sensitive and rapid method to detect 27 beta-thalassemia mutations using pyrosequencing.
  
  
  METHODS
  Pyrosequencing primer pairs and sequencing primers were designed to detect 27 most common beta-thalassemia mutations found in Singapore. Pyrosequencing was performed on 191 DNA samples with known beta-thalassemia mutations isolated from 143 peripheral blood and 48 prenatal samples (seven chorionic villus biopsies, 26 cultured amniocytes, 15 uncultured amniocytes). All mutations were validated with Sanger sequencing.
  
  
  RESULTS
  Pyrosequencing identified 210 alleles with beta-thalassemia mutations and 82 alleles without mutations with 100% sensitivity (lower 95% confidence interval [CI], 97.8%) and 100% specificity (lower 95% CI, 94.4%). All pyrosequences were concordant with Sanger-based sequences. Pyrosequencing was able to detect DNA concentrations as low as 2 ng, obviating the need for cell culture in volume-restricted samples. Sample receipt-to-report assay turnaround times were 16 to 18 h (Sanger sequencing) and 4 to 6 h (pyrosequencing).
  
  
  CONCLUSIONS
  Pyrosequencing is a rapid and sensitive method to detect common beta-thalassemia mutations without the need for cell culture, thus reducing the assay turnaround time.
authors.affiliation: Department of Laboratory Medicine, Molecular Diagnosis Centre, National University Hospital, Singapore, Singapore.
authors.forename: Sherry Sze Yee
authors.initials: SS
authors.lastname: Ho
authors.forename: Pei Tee
authors.initials: PT
authors.lastname: Huan
authors.forename: Gek Har
authors.initials: GH
authors.lastname: Leow
authors.forename: Leng Kee
authors.initials: LK
authors.lastname: Ching
authors.forename: Lily
authors.initials: L
authors.lastname: Chiu
authors.forename: Hai Yang
authors.initials: HY
authors.lastname: Law
authors.forename: Evelyn S C
authors.initials: ES
authors.lastname: Koay
mesh_terms: D000653:Amniotic Fluid / Q000166:cytology; D001210:Asia, Southeastern; D002138:Calibration; D002478:Cells, Cultured; D015193:Chorionic Villi Sampling; D004252:DNA Mutational Analysis / Q000379:methods* / Q000592:standards; D005260:Female; D005820:Genetic Testing / Q000379:methods* / Q000592:standards; D006801:Humans; D009154:Mutation; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods* / Q000592:standards; D017086:beta-Thalassemia / Q000175:diagnosis* / Q000235:genetics*